Abstract

AIMS/HYPOTHESIS:

Our understanding of the transcription factors that control the development and function of rodent islet beta cells is advancing rapidly, yet less is known of the role they play in similar processes in human islets.

METHODS:

To characterise the abundance and regulation of key proteins involved in glucose-regulated insulin secretion in human islets, we examined the expression of MAFA, MAFB, GLUT2 (also known as SLC2A2), βGK (also known as GCK) and PDX1 in isolated, highly purified human islets with an intact insulin secretory pattern. We also assessed these features in islets from two different mouse strains (C57BL/6J and FVB).

RESULTS:

Compared with mouse islets, human islets secreted more insulin at baseline glucose (5.6 mmol/l), but less upon stimulation with high glucose (16.7 mmol/l) or high glucose plus 3-isobutyl-1-methyl-xanthine. Human islets had relatively more MAFB than PDX1 mRNA, while mouse islets had relatively more Pdx1 than Mafb mRNA. However, v-maf musculoaponeurotic fibrosarcoma oncogene homologue (MAF) B protein was found in human islet alpha and beta cells. This is unusual as this regulator is only produced in islet alpha cells in adult mice. The expression of insulin, MAFA, βGK and PDX1 was not glucose-regulated in human islets with an intact insulin secretory pattern.

CONCLUSIONS/INTERPRETATION:

Our results suggest that human islets have a distinctive distribution and function of key regulators of the glucose-stimulated insulin secretion pathway, emphasising the urgent need to understand the processes that regulate human islet beta cell function.

Human islets were isolated by handpicking under microscopic guidance. a An example of a human islet preparation received from an islet isolation facility and stained by dithizone (DTZ) before and (b) after the handpicking procedure. While this example was one of the more impure preparations studied (stated purity 50%), all islet preparations contained ductal and acinar fragments. During culture, acinar fragments rounded up and became similar in size and shape to human islets. These acinar structures could only be distinguished from islets by the lighter brown colour of human islets noted by experienced observers. In contrast, isolated mouse islets were much easier to distinguish from acinar tissue fragments and could readily be identified for handpicking. c An example of mouse islets stained by DTZ after handpicking. Scale bar (a–c) 100 μm. To further evaluate the purity of handpicked islets (d–f), selected islet preparations were processed for cryosections and labelled for insulin (Ins, green), glucagon (Glu, green) and α-amylase (red). The islet cell composition of human and mouse islets was similar to that observed in a previous report []. Note (d) that the size of some acinar fragments (amylase-positive) is similar to that of islets. Adjacent sections (g–i) were labelled for insulin (green), glucagon (red) and somatostatin (Som, blue). Note the difference in islet cell distribution between human and mouse islets. Scale bar (d–i) 100 μm. j High enrichment of human (n = 6) and (k) FVB mouse islet preparations (n = 4) for beta cells vs acinar cells was demonstrated by quantitative RT-PCR

MAFA, PDX1 and MAFB are present in the human islet beta cell population. The percentage of MAFA-, MAFB- or PDX1-producing alpha (glucagon, Glu), beta (insulin, Ins) and delta (somatostatin, Som) cells was determined from the immunohistochemical staining pattern. The intact human pancreas index (a, e, i, m, q) was 55% MAFA+ beta cells (n = 2,935 [total number of analysed cells]), 80% PDX1+ beta cells (n = 1,700), 9% MAFB+ beta cells (n = 1,567) and 40% MAFB+ alpha cells (n = 1,150). MAFA and PDX1 were produced in a larger fraction of beta cells in the mouse pancreas (c, g, k, o, s): 95% MAFA+ beta cells (n = 2,470), 100% PDX1+ beta cells (n = 1,516), 0.4% MAFB+ beta cells (n = 670) and 73% MAFB+ alpha cells (n = 327). e–h Arrows point to Ins+/MAFB+ cells, arrowheads indicate Ins−/MAFB+ cells. Scale bar (a) 50 μm applies to all other panels (b–t). Because, in human islets, beta and non-beta cells are more intermingled, two criteria were used for cell-counting purposes to ensure cell identity. First, an islet cell was deemed positive for nuclear factor or TUNEL only when at least 75% of the nucleus was surrounded by the cytoplasm labelled for a given hormone. Second, if the first criterion was met and there was a gap between the nucleus and the cell cytoplasm, this cell was excluded from the analysis. Thus, it is possible that due to the stringency of our counting procedure, counts of cells doubly positive for a hormone and a transcription factor (especially in human islets) may have been slightly underestimated